The use of intact monoclonal antibodies (MAb), which provide superior binding specificity and affinity for a tumor-associated antigen, have been successfully applied in the area of cancer treatment and diagnosis (A). However, the large size of intact MAbs, their poor bio-distribution and long persistence in the blood pool have limited their clinical applications (B). For example, intact antibodies can exhibit specific accumulation within the tumor area. In biodistribution studies, an inhomogeneous antibody distribution with primary accumulation in the peripheral regions is noted when precisely investigating the tumor. Due to tumor necroses, inhomogeneous antigen distribution and increased interstitial tissue pressure, it is not possible to reach central portions of the tumor with intact antibody constructs. In contrast, smaller antibody fragments show rapid tumor labeling, penetrate deeper into the tumor, and also, are removed relatively rapidly from the bloodstream.
Single chain fragments (scFv) that are derived from the small binding domain of the parent MAb, offer better pharmacokinetics than intact MAbs for clinical application and can target tumor cells more efficiently (C). Single chain fragments can be efficiently engineered from bacteria, however, most engineered scFv have a monovalent structure and show decreased affinity, e.g., a short residence time on a tumor cell, and specificity as compared to their parent MAb ((C(c),D).
In order to increase the affinity and specificity of the scFv for a target antigen, the creation of a multimer, i.e., a multivalent scFv, is desirable. Multivalent antibody constructs such as multibodies (e.g, diabodies, tri-/tetrabodies), and other minibodies offer many advantages in tumor therapy. Recent constructs of multivalent scFv show 102 to 103 times lower off-rate and remarkable increase in binding affinity as compared to a monovalent scFv. Multivalent constructs are advantageous in tumor therapy as a result of improved pharmacokinetic properties and have been further developed for use in tumor therapy (E). They can be used as vehicles for specific accumulation of e.g. cytotoxic substances such as chemotherapeutic agents or radionuclides in a tumor. By suitably selecting the radionuclides, it is possible to destroy tumor cells over a distance of several cell diameters, so that even antigen-negative tumor cells in a tumor area can be covered and poor penetration of antibodies into solid tumors can be compensated at least in part. In spite of these many advantages for multivalent scFvs in cancer therapy, limited methods exist for producing multivalent scFvs.
Currently, most scFv is genetically engineered through bacterial or phase display libraries (E(a-d),E(f),F). In this way, scFv is composed of a heavy chain (VH) and a light chain (VL). The two chains are covalently linked through a polypeptide linker. A multivalent scFv can be prepared by manipulating the length of the polypeptide linker. For example, when the polypeptide linker length is less than five amino acids, the short linker precludes VH-VL association but drives non-covalent dimerization to give a di-scFv. If the length is less than three amino acids, a triabody scFv could be obtained (E(a),E(c),F,G). This method is the most common used method to generate multivalent scFv in spite of the fact that folding is often distorted and specificity is reduced for this type of multivalent scFvs (H).
Alternatively, multivalent scFv can also be prepared by using multimeric affinity reagents such as polyvalent proteins or antibodies. In this way, scFv are anchored on multimers through non-covalent interactions. The main drawback to this method is poor pharmacokinetics, low throughput, and high cost. Additionally, chemical cross-linking using linkers with bi-functional groups has also been investigated but product yields of the cross-linked proteins were limited (I).
In earlier work, the inventors developed a scFv construct against tumor-associated MUC-1 antigen expressed on surface of breast cancer cells (J). In order to better target tumor cells expressing the mucin MUC-1 antigen, a multivalent scFv that can preserve specificity with improved affinity is desirable. Toward this end, the inventors constructed a di-scFv through a melamide-PEG-melamide linker by site-specific PEGylation (I(c)) in low yields (10%-30%).
Alkyne-azide 1,3-dipolar cycloaddition is a highly chemoselective, bioorthogonal (K) chemistry that has been established as an effective chemistry for covalent modification of macromolecules such as proteins (L,M), DNA (N), carbohydrate (O), even virus particle (K,P) and bacterial surfaces (Q). In these cases, a large macromolecule (e.g., protein, DNA, etc.) comprising a reactive group required for the 1,3-dipolar cycloaddition reaction, (i.e., an azide or alkyne) reacts with a small organic molecule comprising the complementary functional group required for the 1,3-dipolar cycloaddition reaction (i.e., an azide or alkyne), to form a 1,2,3-triazole to result in the ligation of a small organic molecule to the large macromolecule.
However, prior to the present invention, alkyne-azide 1,3-dipolar cycloaddition reactions have not been successfully used in the ligation of two large macromolecules each having a single site for the attachment of reactive functional groups (i.e., an azide or an alkyne). It is thought that steric hindrance and low effective concentration of the reacting functional groups are the main impediments to the reaction.
In view of the above, there remains a need in the art for a practical, universal and efficient method to ligate two large macromolecules (e.g., proteins) to produce a conjugated macromolecule, such as a multivalent scFv protein. The present invention fulfills this and other needs.
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